Recombinant engineered bacteria for increasing lincomycin a component and reducing lincomycin b component and its application
A technology of recombinant engineering bacteria and lincomycin, applied in the biological field, can solve problems such as high toxicity, achieve the effects of high titer, simplify the downstream separation and purification process, and improve economic benefits
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Embodiment 1
[0045] A recombinant engineering bacterium that increases component A of lincomycin and reduces component B is constructed by the following method:
[0046] (1) Using the Streptomyces lincoeum genome as a template, design the following primers: use the sequence shown in SEQ ID No.1 as the upstream primer, and use the sequence shown in SEQ ID No.2 as the downstream primer to clone the lmbW gene (GenBank: EU124663.1) ;
[0047] 5'-GCCC AAGCTT CTTCAGACGGACCGAGGTGCC-3' (the underline is the HindIII restriction site); SEQID No.1
[0048] 5'-GCCG GGATCC TCCGCGTGGTTCAGGGCACA-3' (the underline is the BamHI restriction site); SEQ ID No.2
[0049] The 50 μL PCR reaction system is: 10 μL of 5× buffer, 5 μL dNTPs, 5 μL DMSO, 1 μL upstream primer, 1 μL downstream primer, 1 μL genome template, 1 μL polymerase, and make up the rest with purified water.
[0050] The PCR parameters are as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min; 72°C for 10 m...
Embodiment 2
[0059] The second recombinant engineering bacterium that improves the lincomycin A component and reduces the lincomycin B component is constructed by the following method:
[0060] (1) Using the Streptomyces lincoeum genome as a template, design the following primers: use the sequence shown in SEQ ID No.3 as the upstream primer, and use the sequence shown in SEQ ID No.4 as the downstream primer to clone the metK gene (GenBank: KP225159);
[0061] 5'-ACGC AAGCTT GTGTCCCGTCGTCTGTTCAC-3' (the underline is the HindIII restriction site); SEQID No.3
[0062] 5'-GCCG GAATTC GGGGCACCGCGTAGTCGAAGTA-3' (the underline is the EcoRI restriction site); SEQID No.4
[0063] The PCR reaction system and parameter settings are the same as in Example 1 (wherein the primers are different).
[0064] The metK gene fragment was amplified. Using a DNA purification kit, recover the PCR fragment.
[0065] (2) The metK gene was ligated to the pUWL201apr vector by enzyme-cut ligation to construct th...
Embodiment 3
[0070] The third kind of recombinant engineering bacteria that improves the lincomycin A component and reduces the lincomycin B component is constructed by the following method:
[0071] (1) Using the pAP04 vector obtained in Example 1 as a template, using the sequence shown in SEQ ID No.5 as an upstream primer, and using the sequence shown in SEQ ID No.2 as a downstream primer, clone the lmbW gene;
[0072] 5'-GCCG GAATTC GCCCGATGCTAGTCGCGGTTG-3' (underlined is the EcoRI restriction site); SEQ ID No.5 PCR reaction system and parameter settings are the same as in Example 1 (wherein the primers and templates are different).
[0073] The lmbW gene fragment was amplified, and the PCR fragment was recovered with a DNA purification kit.
[0074] (2) The obtained lmbW gene was connected to the pAP05 vector obtained in Example 2 by restriction enzyme digestion, and the pAP06 expression vector in which metK and lmbW were connected in series was constructed.
[0075] The operations ...
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